The Multifaceted Role of Calprotectin (S100A8/A9) in Head and Neck Squamous Cell Carcinoma

Abstract

Calprotectin (S100A8/A9) is formed as a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex. Whereas extracellular calprotectin presents proinflammatory and antimicrobial properties, intracytoplasmic S100A8/A9 appears to play important roles in cell differentiation, cell cycle progression and proliferation, cell migration and survival. While highly upregulated in a variety of cancers, calprotectin is downregulated in head and neck squamous cell carcinoma (HNSCC); loss of S100A8/A9 is associated with increased DNA methylation and poor overall survival rates in HNSCC patients. Using immunohistochemical analysis for S100A8 and S100A9 we found that S100A8/A9 levels gradually decreased during progression of head and neck tumorigenesis from intra-oral premalignant (precancerous) epithelial dysplasia to invasive HNSCC. Furthermore, S100A8/A9 expression positively correlated with the level of squamous differentiation of the primary tumor. To investigate the localization of the calprotectin complex during cell cycle progression, S100A8/A9-expressing human HNSCC cells and immortalized oral keratinocytes were synchronized at G1/S and G2 phases of the cell cycle. During cell division, S100A8/A9 appeared to translocate from the cytoplasm to the microtubule-organizing centers, decorated the mitotic spindles and co-localized with casein kinase II (CK2). Calprotectin nuclear migration is consistent with a role of S100A8/A9 in the control of the G2/M checkpoint. To probe the role of calprotectin in DNA damage responses (DDR), we exposed S100A8/A9-expressing and S100A8/A9-negative carcinoma cells to genotoxic agents. Interestingly, following low doses of X-radiation and incubation with camptothecin, recruitment of the DNA repair regulatory molecules 53BP1 and γH2AX increased significantly in all calprotectin-positive carcinoma cells but failed to increase in calprotectin-negative cancer cells, suggesting impaired DDR in the absence of S100A8/A9. Furthermore, post-radiation DNA fragmentation was more prominent in calprotectin-positive cells as assessed by comet assays. S100A8/A9-negative HNSCC cells also appeared more resistant to cisplatin, while S100A8/A9-expressing carcinoma cells were more sensitive even at lower cisplatin doses. TCGA data indicated that more than 363 apoptosis–related genes were significantly upregulated by S100A8/A9–high HNSCCs compared to S100A8/A9–low neoplasms, including CASP1, -3, -4, -5, -7, -8, -9, -10 and -14. Intracellular calprotectin appeared to promote caspase-mediated DNA fragmentation following radio- and chemotherapy, contributing to S100A8/A9-dependent apoptotic death of carcinomatous cells. In addition, in vitro and ex vivo experiments showed that S100A8/A9 levels were inversely correlated to membranous and cytoplasmic EGFR expression, a negative prognosticator for HNSCC. Calprotectin-associated control of DNA damage responses, post-radiation sensitivity and cisplatin cytotoxicity, and EGFR expression could contribute to the increased overall survival rates of patients with S100A8/A9-high HNSCCs. Our current data further supports the tumor-suppressive role of calprotectin in HNSCC and points to new molecular targets for therapy.